0021-972X/90/7102-0516$0.200/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 71, No. 2 Printed in U.S.A.
Effect of Acute Hypercalcemia on Thyrotropin (TSH) and Triiodothyronine Responses to TSH-Releasing Hormone in Man* C. GILLET, J. CORVILAIN, J. MATTE-HIRIART, D. WILLEMS, AND P. BERGMANN Clinic of Endocrinology and Metabolism, Clinic of Radioimmunochemistry and Nuclear Medicine, Laboratory of Experimental Medicine, Hopital Universitaire Brugmann, Free University of Brussels, Brussels, Belgium
ABSTRACT. In chronic hypercalcemia, basal TSH has been found to be low, with normal serum circulating concentrations of T3 and T4. This observation suggested a potentiation by hypercalcemia of the thyroid secretory response to TSH. The present study was undertaken to assess the possible influence of hypercalcemia on the T3 secretory response to TSH. Since T 3 secretion was studied after stimulation of endogenous TSH by TRH, it was first necessary to find a protocol enabling us to study the effect of calcium on T3 release without affecting TSH secretion. Eighteen subjects underwent two TRH tests, with and without simultaneous calcium infusion, at 2-week interval and in a randomized order. In group A (five subjects) calcium infusion started 1 min after TRH, in group B (five subjects) 10 min after
TRH, and in group C (eight subjects) 20 min after TRH. In groups A and B, TSH secretion was markedly blunted by hypercalcemia. In contrast, when calcium infusion was started 20 min after TRH (group C), the TSH secretion profile was no longer different from that in the control study. However, in this situation the increments of T3 and free T 3 120 and 180 min after TRH were significantly higher when the subjects were rendered hypercalcemic than in the control study. These findings suggest that calcium might act at two different levels, to enhance the thyroid secretory response to TSH and decrease TSH secretion by acting directly on the pituitary gland. Both effects would produce the association of low serum TSH and normal levels of T3 and T4 observed in chronic hypercalcemia. (J Clin Endocrinol Metab 7 1 : 516-519,1990
I
calcium infusion on the thyroid response to endogenous TSH after the administration of TRH. Since acute hypercalcemia is known to blunt the TSH response to TRH when hypercalcemia occurs 2 h before TRH test (2), we, first, had to find an experimental protocol allowing us to dissociate the effect of calcium on serum TSH release after TRH from its possible effect on the thyroid secretory response to TSH. We now describe the influence of the time lag between the occurrence of hypercalcemia and TRH administration on the pituitary secretion of TSH. We also report that in the group of subjects in whom TSH secretion was not affected by hypercalcemia, the increase in T 3 after TRH administration was greater in the presence of a raised serum calcium than in the control study.
N A PREVIOUS study we found that basal serum TSH was lower in patients with primary hyperparathyroidism than in normal subjects (1). However, in these patients circulating T 3 and T4 were normal and remained unchanged after removal of the parathyroid adenoma despite a postoperative rise in circulating TSH within the normal range. In addition, serum TSH was decreased in patients with hypercalcemia of malignancy and increased after correction of hypercalcemia with disphosphonates. This suggested that in patients with primary hyperparathyroidism, hypercalcemia, rather than the elevated circulating PTH concentration, was responsible for the observed low serum TSH. This discrepancy between the low basal TSH and the normal concentration of the circulating thyroid hormones in chronic hypercalcemia raised the possibility of an enhancing effect of calcium on the thyroid secretory response to TSH. To assess that possibility, we studied the effect of
Subjects
Received January 4,1990. Address all correspondence and requests for reprints to: Clementine Gillet, Clinic of Endocrinology and Metabolism, Hopital Universitaire Brugmann, Place Van Gehuchten 4, B-1020 Brussels, Belgium. * This work was supported by the Fonds de la Recherche Scientifique Medicale Beige (Grants 1/5447/885/F and 3/4506189).
Eighteen healthy men, aged 20-30 yr, participated in this study. They were clinically and biochemically euthyroid, and they were not taking any medication. Informed consent was obtained, and the investigation was conducted in accordance with the principles of the Declaration of Helsinki.
Materials and Methods
516
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COMMENTS
517
Study protocol All Fxperiments started at 0800 h after an overnight fast with the subjects resting in a supine position. Indwelling venous cannulae were inserted in both antecubital veins: one for blood sampling, the other for TRH injection and infusion of either 5% glucose or calcium. Blood was drawn for basal determinations of TSH, T3, T4, T4-binding globulin (TBG), and calcium after 60 min of bed rest, and then synthetic TRH (200 ng) was given iv as a single bolus (time zero). To induce hypercalcemia (TRH Ca+), Ca gluconate was given as a bolus (3 mg/kg BW elemental Ca), followed immediately by a constant infusion of Ca gluconate in 5% glucose at a rate of 50 mL/hour (3.6 mg elemental Ca/kg BW-h) until 180 min after TRH. The subjects were subdivided into three groups: group A (n = 5), calcium bolus was given 1 min after TRH; group B (n = 5), calcium bolus was given 10 min after TRH; and group C (n = 8), calcium bolus was given 20 min after TRH. In the control study (TRH Ca-), instead of calcium gluconate a 5% glucose solution was infused at a constant rate throughout the study. Each subject had two TRH tests at 2week intervals, one with (TRH Ca+) and one without (TRH Ca—) calcium administration. The order of the tests was randomized. Blood samples were drawn at 0, 20, 25, 30, 60, and 120 min for TSH, at 0, 60,120, and 180 min for serum T3, and at 0,120, and 180 min for serum free T 3 (FT3). Serum calcium was measured every 5 min for the first 30 min after TRH administration, and then at 60, 120, and 180 min. Assay procedures T3, T 4 (Amerlex, Amersham, United Kingdom), and TBG (Behring, Marburg, West Germany) were measured by RIA. FT 3 was measured by a one-step method using an analog as tracer (Amerlex). Serum TSH was measured using an ultrasensitive immunoradiometric assay (Behring), with a lower detection limit of 0.05 mU/L, an intraassay precision of 5.5%, and an interassay precision of 8%. Serum calcium was measured by a colorimetric method using a cresolphtalein complexone. The normal ranges were: FT3, 3.3-8.2 pmol/L; T3, 0.92-2.62 nmol/ L; TSH, 0.1-5 mU/L; and serum calcium, 2.28-2.55 mmol/L.
Results Baseline serum concentrations of TSH, T4, T3, TBG, and calcium were not significantly different in TRH Ca— and TRH Ca+ tests (Table 1). During the TRH Ca+ test, serum calcium was maintained between 3.0 and 3.25 mmol/L (Fig. 1). Hypercalcemia was comparable in groups A, B, and C. As shown in Fig. 1, TSH secretion after TRH, expressed as A max TSH, was significantly blunted by hypercalcemia when it was induced 1 (group A) or 10 (group B) min after TRH (P < 0.01). The net secretory area (AUC) was 408 ± 74 mU/L • 120 min (TRH Ca+) vs. 668 ± 133 mU/L-120 min (TRH Ca-) in group A (P < 0.01) and 285 ± 23 mU/L-120 min (TRH Ca+) vs. 573 ± 147 mU/L-120 min (TRH Ca-) in group B (P < 0.01). When calcium was given 20 min after TRH (group C), TSH secretion was identical during TRH C a - and TRH Ca+ test (Fig. 1). Moreover, the AUCs were not significantly different: 633 ± 74 mU/L-120 min (TRH Ca+) vs. 635 ± 85 mU/L-120 min (TRH Ca-). Therefore, the effect of hypercalcemia on the T 3 secretory response to TRH was analyzed in this group only. In the eight subjects in whom calcium was started 20 min after TRH, the mean curves of T 3 obtained in the presence or absence of hypercalcemia were significantly different, as assessed by analysis of variance (P < 0.01 for T3; Fig. 2). The increment of T 3 at 120 (P < 0.01) and 180 min (P < 0.05) and the cumulative increment of T 3 (1.79 ± 0.15 vs. 1.26 ± 0.22 nmol/L; P < 0.01) were significantly higher when the subjects had received calcium (Table 2). These results seen with total T 3 were further confirmed by FT 3 measurements. The mean curves of FT 3 obtained in the presence or absence of hypercalcemia were significantly different as assessed by analysis of variance (P = 0.01, Fig. 2). The increments of FT 3 at 120 (P < 0.05) and 180 min (P < 0.05) and the cumulative increment of FT 3 (5.9 ± 0.6 vs. 4.0 ± 0.5 pmol/L; P < 0.01) after TRH were significantly higher during calcium infusion (Table 2).
Statistical analysis Data are presented as the mean ± SEM. The maximum increment of TSH (A max TSH) was calculated for each subject as the difference between baseline and peak serum level of TSH; the net TSH secretory area under the curve (AUC) was calculated by the trapezoidal method. Increment above basal was calculated at different time intervals for T 3 (A T3) and FT 3 (A FT3). The cumulative T 3 and FT 3 increment was obtained by summing A T 3 at 60, 120, and 180 min and A FT 3 at 120 and 180 min. Analysis of variance for repeated sampling was used to assess the effects of endogenous TSH on T3 and FT3. Significance of differences was assessed by variance analysis and Wilcoxon matched pairs tests.
Discussion Our previous observation (1) of normal serum T4 and T 3 concentrations in the presence of a low serum TSH TABLE 1. Mean baseline values of serum calcium, TSH, and thyroid hormones in 18 subjects before the TRH test, with (TRH Ca+) or without (TRH Ca-) calcium administration
Ca (mmol/L) TSH (mU/L) T3 (nmol/L) T4 (nmol/L) TBG (mg/L)
TRH Ca(n = 18)
TRH Ca+ (n = 18)
2.32 ± 0.02 1.41 ± 0.14 1.85 ± 0.08 89.5 ± 3.0 15.1 ± 0.9
2.32 ± 0.02 1.39 ± 0.16 1.90 ± 0.09 90.2 ± 3.90 14.7 ± 0.70
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JCE & M • 1990 Vol 71 • No 2
COMMENTS
518
TRH 200 /ig 12 -i 11 10
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FIG. 1. Mean (±SE) serum TSH in response to TRH injection (200 fig, iv; upper panel) in the three groups of normal subjects during control studies (O- -O) and when calcium was given (•—•) 1 min (A; n = 5), 10 min (B; n = 5), and 20 min (C; n = 8) after TRH. Serum calcium at different time intervals without (O- -O) or with (•—•) calcium infusion {lower panel) is shown. **, Serum TSH concentration significantly different from the control, P < 0.01.
level in chronic hypercalcemia prompted us to investigate the effect of calcium on the thyroid sensitivity to TSH. Since adverse reactions after the injection of bovine TSH have been reported, particularly if given more than once, we studied the effect of calcium infusion on T 3 secretion after an increase in endogenous TSH, obtained through TRH administration. As hypercalcemia was known to inhibit TRH-induced TSH secretion (2), we first had to find an experimental protocol allowing us to dissociate the effect of a raised serum calcium level on the pituitary gland from its possible influence on the thyroid response to TSH. Our study demonstrated that the effect of Ca on TSH secretion was rapid. Even when given 10 min after TRH, calcium significantly decreased the serum TSH concentration attained at 20 min. The finding of a decreased TSH response to TRH after induction of moderate hypercalcemia seems inconsistent with available in vitro data. In mouse thyrotropic tumor cells (TtT) the TRH action on TSH secretion involves an increase of intracellular Ca2+ resulting from the mobilization of cellular Ca2+ stores and from an enhancement of Ca2+ influx that increases with extracellular Ca2+ concentration ranging from 10-3000 /xM (3). Accordingly, TRH-induced TSH secretion was shown to increase in the TtT cells for extracellular calcium concentrations ranging from 31500 nM (4). Concentrations corresponding to the hypercalcemia achieved in our study was perhaps not explored in this latter in vitro study. An explanation for the paradoxical inhibitory effect of hypercalcemia on TSH secretion in vivo is that hyper-
calcemia could blunt the pituitary release of TSH by stimulating the release of dopamine from the hypothalamus. The fact that pretreatment with metoclopramide, an antagonist of dopamine, restores a normal TSH response to TRH in subjects with exogenous hypercalcemia and does not modify it in controls (5) lends experimental support for this hypothesis. When calcium infusion was started 20 min after TRH administration, the TSH secretion profile was comparable to that in the absence of hypercalcemia. This protocol, which did not affect TSH secretion, was, therefore, used to assess the thyroid response to TRH in the presence of acute hypercalcemia. In these experiments we observed that, as assessed by the changes in serum T3, the response of the thyroid gland after an increase in endogenous TSH was greater during the TRH Ca+ test than during TRH C a - test. A possible explanation for a greater rise in serum T 3 with calcium infusion might be that calcium would increase binding of T 3 to carrier proteins, but since the same observation was made with FT3, this explanation seems unlikely. A calcium-induced increase of the bioactivity/immunoactivity ratio of circulating TSH (6) also appears doubtful, since most of the secretory response of TSH to TRH had been achieved before calcium administration. As the half-life of T 3 is between 24-30 h (7), a decrease in T 3 catabolism alone also could not account for the 50% difference in A T3. The present data seem to suggest that calcium acts at the thyroid level. In the absence of convincing evidence favoring a direct effect of TRH on
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 17:16 For personal use only. No other uses without permission. . All rights reserved.
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anisms in the thyroid cell (8, 9). Indeed, it has been shown that TSH can activate the phosphatidyl inositol cascade (10, 11) and yield DG and IP3, which, in turn, would increase intracellular calcium by releasing nonmitochondrial calcium stores and opening calcium channels allowing calcium influx from extracellular medium as in other cells (12). It is possible that extracellular calcium could be involved in this pathway. On the other hand, hypercalcemia could also affect the number of TSH receptors or their affinity for the hormone itself. If our findings could be extended to a state of chronic hypercalcemia, then a higher sensitivity of the thyroid to TSH could be the first event explaining the paradoxical association of low serum TSH and normal thyroid hormone concentrations observed in primary hyperparathyroidism and in the hypercalcemia of malignancy. However, the rapid effect of hypercalcemia on the TSH response to TRH suggests that calcium also decreases serum TSH by a direct action on the pituitary gland, probably by inhibiting TSH secretion. Hypercalcemia would then act at two different levels, and an increased response of the thyroid to TSH would maintain normal serum thyroid hormone concentrations in spite of a low serum TSH level.
TIME AFTER TRH INJECTION
FlG. 2. Left, Mean (±SE) serum AT3 and AFT3 concentrations after TRH (200 fig, iv) in eight normal subjects when hypercalcemia was induced 20 min after TRH (TRH Ca+; • — • ) and in the control study (TRH Ca-; O- -O). Right, Mean (±SE) sums of 60, 120, and 180 min A T3 and 120 and 180 min A FT3 after TRH in the control study (Ca-) and when calcium was given 20 min after TRH (Ca+). The increases in T 3 and FT3 were significantly greater during hypercalcemia than in the control study. *, P < 0.05; **, P < 0.01. TABLE 2. A T3 and A FT 3 at different time intervals after TRH administration in eight subjects when calcium was given 20 min after TRH (TRH Ca+) and in the control study (TRH Ca-)
A T3 (nmol/L) 60 min 120 min 180 min A FT3 (pmol/L) 120 min 180 min
TRH Cadi =8)
TRH Ca+ (n = 8)
0.20 ± 0.06 0.48 ± 0.08 0.55 ± 0.08
0.28 ± 0.04 0.72 ± 0.08° 0.80 ± 0.08"
1.5 ± 0.2 2.48 ± 0.0
2.6 ± 0.37" 3.34 ± 0.40"
Values are the mean ± SE. P < 0.01 compared to control. b P< 0.05 compared to control. a
thyroid hormone secretion, our results strongly suggest that calcium enhances the thyroid sensitivity to TSH. The precise way in which calcium influences the thyroid cell remains speculative. It is noteworthy that besides cAMP, a well known intracellular second messenger, calcium itself could act as one of the transduction mech-
References 1. Gillet C, Bergmann P, Francois D, Body JJ, Corvilain J. Low basal thyrotropin with normal thyroid function in primary hyperparathyroidism. Acta Endocrinol (Copenh). 1989;121(Suppl 5):638-42. 2. Dudczak R, Waldhausl WK, Bratusch-Marrain P. Effect of disodium EDTA and calcium infusion on prolactin and thyrotropin responses to thyrotropin releasing hormone in healthy man. J Clin Endocrinol Metab. 1983;56:603-7. 3. Geras-Raaka E, Gershengorn MC. Measurement of changes in cellular calcium metabolism in response to thyrotropin-releasing hormone. Methods Enzymol. 1987;141:36-53. 4. Geras E, Gershengorn MC. Evidence that TRH stimulates secretion of TSH by two calcium mediated mechanism. Am J Physiol. 1982;242:E1O9-14. 5. Rojdmark S, Andersson DEH. Influence of metoclopramide on calcium inhibition of thyrotropin response to thyrotropin releasing hormone. J Clin Endocrinol Metab. 1982;54:998-1001. 6. Dahlberg PA, Petrick PA, Nissim M, Menezes-Ferreira MM, Weintraub BD. Intrinsic bioactivity of thyrotropin in human serum is inversely correlated with thyroid hormone concentrations. J Clin Invest. 1987;79:1388-93. 7. Werner S. The thyroid: a fundamental and clinical text, 5th ed. Philadelphia: Lippencott; 1980;139. 8. Rasmussen H. The calcium messenger system. N Engl J Med. 1986;314:1094-101. 9. Rani SCS, Schilling WP, Field JB. Intracellular Ca2+ mobilization by thyrotropin, carbachol, and adenosine triphosphate in dog thyroid cells. Endocrinology. 1989;125:1889-97. 10. Laurent E, Mockel J, Van Sande J, Graff I, Dumont JE. Dual activation by thyrotropin of the phospholipase C and cAMP cascades in human thyroid. Mol Cell Endocrinol. 1987;52:273-8. 11. Ikeda M, Deery WJ, Ferdows MS, Nielsen TB, Field JB. Role of cellular Ca2+ in phosphorylation of 21K and 19K polypeptides in cultured thyroid cells: effects of phorbol ester, trifluoperazine, and 8-diethylamino-octyl-3,4,5-trimethoxy-benzoate hydrochloride. Endocrinology. 1987;121:175-81. 12. Berridge MJ, Irvine RF. Inositol phosphates and cell signalling. Nature. 1989;341:197-205.
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Vol. 71, No. 2 Printed in U.S.A.
Effect of Acute Hypercalcemia on Thyrotropin (TSH) and Triiodothyronine Responses to TSH-Releasing Hormone in Man* C. GILLET, J. CORVILAIN, J. MATTE-HIRIART, D. WILLEMS, AND P. BERGMANN Clinic of Endocrinology and Metabolism, Clinic of Radioimmunochemistry and Nuclear Medicine, Laboratory of Experimental Medicine, Hopital Universitaire Brugmann, Free University of Brussels, Brussels, Belgium
ABSTRACT. In chronic hypercalcemia, basal TSH has been found to be low, with normal serum circulating concentrations of T3 and T4. This observation suggested a potentiation by hypercalcemia of the thyroid secretory response to TSH. The present study was undertaken to assess the possible influence of hypercalcemia on the T3 secretory response to TSH. Since T 3 secretion was studied after stimulation of endogenous TSH by TRH, it was first necessary to find a protocol enabling us to study the effect of calcium on T3 release without affecting TSH secretion. Eighteen subjects underwent two TRH tests, with and without simultaneous calcium infusion, at 2-week interval and in a randomized order. In group A (five subjects) calcium infusion started 1 min after TRH, in group B (five subjects) 10 min after
TRH, and in group C (eight subjects) 20 min after TRH. In groups A and B, TSH secretion was markedly blunted by hypercalcemia. In contrast, when calcium infusion was started 20 min after TRH (group C), the TSH secretion profile was no longer different from that in the control study. However, in this situation the increments of T3 and free T 3 120 and 180 min after TRH were significantly higher when the subjects were rendered hypercalcemic than in the control study. These findings suggest that calcium might act at two different levels, to enhance the thyroid secretory response to TSH and decrease TSH secretion by acting directly on the pituitary gland. Both effects would produce the association of low serum TSH and normal levels of T3 and T4 observed in chronic hypercalcemia. (J Clin Endocrinol Metab 7 1 : 516-519,1990
I
calcium infusion on the thyroid response to endogenous TSH after the administration of TRH. Since acute hypercalcemia is known to blunt the TSH response to TRH when hypercalcemia occurs 2 h before TRH test (2), we, first, had to find an experimental protocol allowing us to dissociate the effect of calcium on serum TSH release after TRH from its possible effect on the thyroid secretory response to TSH. We now describe the influence of the time lag between the occurrence of hypercalcemia and TRH administration on the pituitary secretion of TSH. We also report that in the group of subjects in whom TSH secretion was not affected by hypercalcemia, the increase in T 3 after TRH administration was greater in the presence of a raised serum calcium than in the control study.
N A PREVIOUS study we found that basal serum TSH was lower in patients with primary hyperparathyroidism than in normal subjects (1). However, in these patients circulating T 3 and T4 were normal and remained unchanged after removal of the parathyroid adenoma despite a postoperative rise in circulating TSH within the normal range. In addition, serum TSH was decreased in patients with hypercalcemia of malignancy and increased after correction of hypercalcemia with disphosphonates. This suggested that in patients with primary hyperparathyroidism, hypercalcemia, rather than the elevated circulating PTH concentration, was responsible for the observed low serum TSH. This discrepancy between the low basal TSH and the normal concentration of the circulating thyroid hormones in chronic hypercalcemia raised the possibility of an enhancing effect of calcium on the thyroid secretory response to TSH. To assess that possibility, we studied the effect of
Subjects
Received January 4,1990. Address all correspondence and requests for reprints to: Clementine Gillet, Clinic of Endocrinology and Metabolism, Hopital Universitaire Brugmann, Place Van Gehuchten 4, B-1020 Brussels, Belgium. * This work was supported by the Fonds de la Recherche Scientifique Medicale Beige (Grants 1/5447/885/F and 3/4506189).
Eighteen healthy men, aged 20-30 yr, participated in this study. They were clinically and biochemically euthyroid, and they were not taking any medication. Informed consent was obtained, and the investigation was conducted in accordance with the principles of the Declaration of Helsinki.
Materials and Methods
516
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COMMENTS
517
Study protocol All Fxperiments started at 0800 h after an overnight fast with the subjects resting in a supine position. Indwelling venous cannulae were inserted in both antecubital veins: one for blood sampling, the other for TRH injection and infusion of either 5% glucose or calcium. Blood was drawn for basal determinations of TSH, T3, T4, T4-binding globulin (TBG), and calcium after 60 min of bed rest, and then synthetic TRH (200 ng) was given iv as a single bolus (time zero). To induce hypercalcemia (TRH Ca+), Ca gluconate was given as a bolus (3 mg/kg BW elemental Ca), followed immediately by a constant infusion of Ca gluconate in 5% glucose at a rate of 50 mL/hour (3.6 mg elemental Ca/kg BW-h) until 180 min after TRH. The subjects were subdivided into three groups: group A (n = 5), calcium bolus was given 1 min after TRH; group B (n = 5), calcium bolus was given 10 min after TRH; and group C (n = 8), calcium bolus was given 20 min after TRH. In the control study (TRH Ca-), instead of calcium gluconate a 5% glucose solution was infused at a constant rate throughout the study. Each subject had two TRH tests at 2week intervals, one with (TRH Ca+) and one without (TRH Ca—) calcium administration. The order of the tests was randomized. Blood samples were drawn at 0, 20, 25, 30, 60, and 120 min for TSH, at 0, 60,120, and 180 min for serum T3, and at 0,120, and 180 min for serum free T 3 (FT3). Serum calcium was measured every 5 min for the first 30 min after TRH administration, and then at 60, 120, and 180 min. Assay procedures T3, T 4 (Amerlex, Amersham, United Kingdom), and TBG (Behring, Marburg, West Germany) were measured by RIA. FT 3 was measured by a one-step method using an analog as tracer (Amerlex). Serum TSH was measured using an ultrasensitive immunoradiometric assay (Behring), with a lower detection limit of 0.05 mU/L, an intraassay precision of 5.5%, and an interassay precision of 8%. Serum calcium was measured by a colorimetric method using a cresolphtalein complexone. The normal ranges were: FT3, 3.3-8.2 pmol/L; T3, 0.92-2.62 nmol/ L; TSH, 0.1-5 mU/L; and serum calcium, 2.28-2.55 mmol/L.
Results Baseline serum concentrations of TSH, T4, T3, TBG, and calcium were not significantly different in TRH Ca— and TRH Ca+ tests (Table 1). During the TRH Ca+ test, serum calcium was maintained between 3.0 and 3.25 mmol/L (Fig. 1). Hypercalcemia was comparable in groups A, B, and C. As shown in Fig. 1, TSH secretion after TRH, expressed as A max TSH, was significantly blunted by hypercalcemia when it was induced 1 (group A) or 10 (group B) min after TRH (P < 0.01). The net secretory area (AUC) was 408 ± 74 mU/L • 120 min (TRH Ca+) vs. 668 ± 133 mU/L-120 min (TRH Ca-) in group A (P < 0.01) and 285 ± 23 mU/L-120 min (TRH Ca+) vs. 573 ± 147 mU/L-120 min (TRH Ca-) in group B (P < 0.01). When calcium was given 20 min after TRH (group C), TSH secretion was identical during TRH C a - and TRH Ca+ test (Fig. 1). Moreover, the AUCs were not significantly different: 633 ± 74 mU/L-120 min (TRH Ca+) vs. 635 ± 85 mU/L-120 min (TRH Ca-). Therefore, the effect of hypercalcemia on the T 3 secretory response to TRH was analyzed in this group only. In the eight subjects in whom calcium was started 20 min after TRH, the mean curves of T 3 obtained in the presence or absence of hypercalcemia were significantly different, as assessed by analysis of variance (P < 0.01 for T3; Fig. 2). The increment of T 3 at 120 (P < 0.01) and 180 min (P < 0.05) and the cumulative increment of T 3 (1.79 ± 0.15 vs. 1.26 ± 0.22 nmol/L; P < 0.01) were significantly higher when the subjects had received calcium (Table 2). These results seen with total T 3 were further confirmed by FT 3 measurements. The mean curves of FT 3 obtained in the presence or absence of hypercalcemia were significantly different as assessed by analysis of variance (P = 0.01, Fig. 2). The increments of FT 3 at 120 (P < 0.05) and 180 min (P < 0.05) and the cumulative increment of FT 3 (5.9 ± 0.6 vs. 4.0 ± 0.5 pmol/L; P < 0.01) after TRH were significantly higher during calcium infusion (Table 2).
Statistical analysis Data are presented as the mean ± SEM. The maximum increment of TSH (A max TSH) was calculated for each subject as the difference between baseline and peak serum level of TSH; the net TSH secretory area under the curve (AUC) was calculated by the trapezoidal method. Increment above basal was calculated at different time intervals for T 3 (A T3) and FT 3 (A FT3). The cumulative T 3 and FT 3 increment was obtained by summing A T 3 at 60, 120, and 180 min and A FT 3 at 120 and 180 min. Analysis of variance for repeated sampling was used to assess the effects of endogenous TSH on T3 and FT3. Significance of differences was assessed by variance analysis and Wilcoxon matched pairs tests.
Discussion Our previous observation (1) of normal serum T4 and T 3 concentrations in the presence of a low serum TSH TABLE 1. Mean baseline values of serum calcium, TSH, and thyroid hormones in 18 subjects before the TRH test, with (TRH Ca+) or without (TRH Ca-) calcium administration
Ca (mmol/L) TSH (mU/L) T3 (nmol/L) T4 (nmol/L) TBG (mg/L)
TRH Ca(n = 18)
TRH Ca+ (n = 18)
2.32 ± 0.02 1.41 ± 0.14 1.85 ± 0.08 89.5 ± 3.0 15.1 ± 0.9
2.32 ± 0.02 1.39 ± 0.16 1.90 ± 0.09 90.2 ± 3.90 14.7 ± 0.70
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JCE & M • 1990 Vol 71 • No 2
COMMENTS
518
TRH 200 /ig 12 -i 11 10
9-1 8 7 •
65 4 3 2
1 0
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120
TIME (MIN)
FIG. 1. Mean (±SE) serum TSH in response to TRH injection (200 fig, iv; upper panel) in the three groups of normal subjects during control studies (O- -O) and when calcium was given (•—•) 1 min (A; n = 5), 10 min (B; n = 5), and 20 min (C; n = 8) after TRH. Serum calcium at different time intervals without (O- -O) or with (•—•) calcium infusion {lower panel) is shown. **, Serum TSH concentration significantly different from the control, P < 0.01.
level in chronic hypercalcemia prompted us to investigate the effect of calcium on the thyroid sensitivity to TSH. Since adverse reactions after the injection of bovine TSH have been reported, particularly if given more than once, we studied the effect of calcium infusion on T 3 secretion after an increase in endogenous TSH, obtained through TRH administration. As hypercalcemia was known to inhibit TRH-induced TSH secretion (2), we first had to find an experimental protocol allowing us to dissociate the effect of a raised serum calcium level on the pituitary gland from its possible influence on the thyroid response to TSH. Our study demonstrated that the effect of Ca on TSH secretion was rapid. Even when given 10 min after TRH, calcium significantly decreased the serum TSH concentration attained at 20 min. The finding of a decreased TSH response to TRH after induction of moderate hypercalcemia seems inconsistent with available in vitro data. In mouse thyrotropic tumor cells (TtT) the TRH action on TSH secretion involves an increase of intracellular Ca2+ resulting from the mobilization of cellular Ca2+ stores and from an enhancement of Ca2+ influx that increases with extracellular Ca2+ concentration ranging from 10-3000 /xM (3). Accordingly, TRH-induced TSH secretion was shown to increase in the TtT cells for extracellular calcium concentrations ranging from 31500 nM (4). Concentrations corresponding to the hypercalcemia achieved in our study was perhaps not explored in this latter in vitro study. An explanation for the paradoxical inhibitory effect of hypercalcemia on TSH secretion in vivo is that hyper-
calcemia could blunt the pituitary release of TSH by stimulating the release of dopamine from the hypothalamus. The fact that pretreatment with metoclopramide, an antagonist of dopamine, restores a normal TSH response to TRH in subjects with exogenous hypercalcemia and does not modify it in controls (5) lends experimental support for this hypothesis. When calcium infusion was started 20 min after TRH administration, the TSH secretion profile was comparable to that in the absence of hypercalcemia. This protocol, which did not affect TSH secretion, was, therefore, used to assess the thyroid response to TRH in the presence of acute hypercalcemia. In these experiments we observed that, as assessed by the changes in serum T3, the response of the thyroid gland after an increase in endogenous TSH was greater during the TRH Ca+ test than during TRH C a - test. A possible explanation for a greater rise in serum T 3 with calcium infusion might be that calcium would increase binding of T 3 to carrier proteins, but since the same observation was made with FT3, this explanation seems unlikely. A calcium-induced increase of the bioactivity/immunoactivity ratio of circulating TSH (6) also appears doubtful, since most of the secretory response of TSH to TRH had been achieved before calcium administration. As the half-life of T 3 is between 24-30 h (7), a decrease in T 3 catabolism alone also could not account for the 50% difference in A T3. The present data seem to suggest that calcium acts at the thyroid level. In the absence of convincing evidence favoring a direct effect of TRH on
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COMMENTS TRH 200 fig
Ca 0.9 -
|
1.8 1.5 1.2 0.9 -0.6 -0.3 •0
0.6-
c
w
0.3 -
^
0 0 20
60
120
180 MIN
Ca" Ca+
0 20
60
120
180 MIN
Ca- Ca+
54321 0-
519
anisms in the thyroid cell (8, 9). Indeed, it has been shown that TSH can activate the phosphatidyl inositol cascade (10, 11) and yield DG and IP3, which, in turn, would increase intracellular calcium by releasing nonmitochondrial calcium stores and opening calcium channels allowing calcium influx from extracellular medium as in other cells (12). It is possible that extracellular calcium could be involved in this pathway. On the other hand, hypercalcemia could also affect the number of TSH receptors or their affinity for the hormone itself. If our findings could be extended to a state of chronic hypercalcemia, then a higher sensitivity of the thyroid to TSH could be the first event explaining the paradoxical association of low serum TSH and normal thyroid hormone concentrations observed in primary hyperparathyroidism and in the hypercalcemia of malignancy. However, the rapid effect of hypercalcemia on the TSH response to TRH suggests that calcium also decreases serum TSH by a direct action on the pituitary gland, probably by inhibiting TSH secretion. Hypercalcemia would then act at two different levels, and an increased response of the thyroid to TSH would maintain normal serum thyroid hormone concentrations in spite of a low serum TSH level.
TIME AFTER TRH INJECTION
FlG. 2. Left, Mean (±SE) serum AT3 and AFT3 concentrations after TRH (200 fig, iv) in eight normal subjects when hypercalcemia was induced 20 min after TRH (TRH Ca+; • — • ) and in the control study (TRH Ca-; O- -O). Right, Mean (±SE) sums of 60, 120, and 180 min A T3 and 120 and 180 min A FT3 after TRH in the control study (Ca-) and when calcium was given 20 min after TRH (Ca+). The increases in T 3 and FT3 were significantly greater during hypercalcemia than in the control study. *, P < 0.05; **, P < 0.01. TABLE 2. A T3 and A FT 3 at different time intervals after TRH administration in eight subjects when calcium was given 20 min after TRH (TRH Ca+) and in the control study (TRH Ca-)
A T3 (nmol/L) 60 min 120 min 180 min A FT3 (pmol/L) 120 min 180 min
TRH Cadi =8)
TRH Ca+ (n = 8)
0.20 ± 0.06 0.48 ± 0.08 0.55 ± 0.08
0.28 ± 0.04 0.72 ± 0.08° 0.80 ± 0.08"
1.5 ± 0.2 2.48 ± 0.0
2.6 ± 0.37" 3.34 ± 0.40"
Values are the mean ± SE. P < 0.01 compared to control. b P< 0.05 compared to control. a
thyroid hormone secretion, our results strongly suggest that calcium enhances the thyroid sensitivity to TSH. The precise way in which calcium influences the thyroid cell remains speculative. It is noteworthy that besides cAMP, a well known intracellular second messenger, calcium itself could act as one of the transduction mech-
References 1. Gillet C, Bergmann P, Francois D, Body JJ, Corvilain J. Low basal thyrotropin with normal thyroid function in primary hyperparathyroidism. Acta Endocrinol (Copenh). 1989;121(Suppl 5):638-42. 2. Dudczak R, Waldhausl WK, Bratusch-Marrain P. Effect of disodium EDTA and calcium infusion on prolactin and thyrotropin responses to thyrotropin releasing hormone in healthy man. J Clin Endocrinol Metab. 1983;56:603-7. 3. Geras-Raaka E, Gershengorn MC. Measurement of changes in cellular calcium metabolism in response to thyrotropin-releasing hormone. Methods Enzymol. 1987;141:36-53. 4. Geras E, Gershengorn MC. Evidence that TRH stimulates secretion of TSH by two calcium mediated mechanism. Am J Physiol. 1982;242:E1O9-14. 5. Rojdmark S, Andersson DEH. Influence of metoclopramide on calcium inhibition of thyrotropin response to thyrotropin releasing hormone. J Clin Endocrinol Metab. 1982;54:998-1001. 6. Dahlberg PA, Petrick PA, Nissim M, Menezes-Ferreira MM, Weintraub BD. Intrinsic bioactivity of thyrotropin in human serum is inversely correlated with thyroid hormone concentrations. J Clin Invest. 1987;79:1388-93. 7. Werner S. The thyroid: a fundamental and clinical text, 5th ed. Philadelphia: Lippencott; 1980;139. 8. Rasmussen H. The calcium messenger system. N Engl J Med. 1986;314:1094-101. 9. Rani SCS, Schilling WP, Field JB. Intracellular Ca2+ mobilization by thyrotropin, carbachol, and adenosine triphosphate in dog thyroid cells. Endocrinology. 1989;125:1889-97. 10. Laurent E, Mockel J, Van Sande J, Graff I, Dumont JE. Dual activation by thyrotropin of the phospholipase C and cAMP cascades in human thyroid. Mol Cell Endocrinol. 1987;52:273-8. 11. Ikeda M, Deery WJ, Ferdows MS, Nielsen TB, Field JB. Role of cellular Ca2+ in phosphorylation of 21K and 19K polypeptides in cultured thyroid cells: effects of phorbol ester, trifluoperazine, and 8-diethylamino-octyl-3,4,5-trimethoxy-benzoate hydrochloride. Endocrinology. 1987;121:175-81. 12. Berridge MJ, Irvine RF. Inositol phosphates and cell signalling. Nature. 1989;341:197-205.
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